The present invention generally relates to the chemistry of the attachment of oligonucleotides to solid supports. More particularly the present invention relates to linking oligonucleotides to solid supports through a Schiff base type covalent linkage for capture and detection of single- and double stranded DNA and RNA targets.
The detection and quantification of very small quantifies of nucleic acids plays an important role in the biological, forensic and medical sciences. Typically nucleic acids in samples are detected by hybridization to a complementary oligonucleotide containing more than 8 contiguous nucleotides. To provide a signal proportional to the target-oligonucleotide hybrid, typically either the target or the second probe contains a signal generating label, such as a radioactive-, fluorescent-, chemiluminescent-moiety or an enzyme (such as horseradish peroxidase) that through its catalytic activity yields a detectable product. The prior art is well developed in this regard and numerous methods are available for the detection and quantification of signal in the nucleic acid field.
Following the hybridization of the capturing and labeled oligonucleotide to the target nucleic acid it is necessary to separate the signal generating duplex from unreacted target and labeled oligonucleotide. This can usually be accomplished because either the target, or more typically the capturing oligonucleotide has been immobilized on a solid support thereby allowing the isolation of the hybrid free from unhybridized molecules. In a xe2x80x9csandwich assayxe2x80x9d variation, an oligonucleotide is immobilized to a solid support and is used to capture a target. The captured target is detected by hybridization with a second labeled oligonucleotide, that has a different sequence than the capturing oligonucleotide.
Numerous types of solid supports suitable for immobilizing oligonucleotides are known in the art. These include nylon, nitrocelluose, activated agarose, diazotized cellulose, latex particles, plastic, polystyrene, glass and polymer coated surfaces. These solid supports are used in many formats such as membranes, microtiter plates, beads, probes, dipsticks etc. A wide variety of chemical procedures are known to covalently link oligonucleotides directly or through a linker to these solid supports. Of particular interest as background to the present invention is the use of glass and nylon surfaces in the preparation of DNA microarrays which have been described in recent years (Ramsay, Nat. Biotechnol., 16: 40-4 (1998)). The journal Nature Genetics has published a special supplement describing the utility and limitations of microarrays (Nat.Genet., 21(1): 1-60 (1999).
Typically the use of any solid support requires the presence of a nucleophilic group to react with an oligonucleotide that must contain a xe2x80x9creactive groupxe2x80x9d capable of reacting with the nucleophilic group. Alternatively, a xe2x80x9creactive groupxe2x80x9d is present or is introduced into the solid support to react with a nucleophile present in or attached to the oligonucleotide. Suitable nucleophilic groups or moieties include hydroxyl, sulfhydryl, amino and activated carboxyl groups, while the groups capable of reacting with these and other nucleophiles (reactive groups) include dichlorotriazinyl, alkylepoxy, maleimido, bromoacetyl goups and others. Chemical procedures to introduce the nucleophilic or the reactive groups on to solid support are known in the art, they include procedures to activate nylon (U.S. Pat. No. 5,514,785), glass (Rodgers et al., Anal. Biochem., 23-30 (1999)), agarose (Highsmith et al., J., Biotechniques 12: 418-23 (1992) and polystyrene (Gosh et al., Nuc. Acid Res., 15: 5353-5372 (1987)). Dependent on the presence of either a reactive or nucleophilic groups on the solid support and oligonucleotide, coupling can either be performed directly or with bifunctional reagents. Bifunctional and coupling reagents are well known in the art and many are available from commercial sources.
Of special interest as background to the present invention is the procedure described by Kremsky et al. (Nuc.Acid Res., 15: 2891-2909 (1987)) for the preparation of a 16-mer oligonucleotide containing a 6 carbon carboxylic acid linker on the 5xe2x80x2-end. This product was synthesized using the appropriate phosphoramidites on a standard synthesizer. The acid was then reacted with 3-amino-1,2-propanediol in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to yield a stable diol. The diol was oxidized to the aliphatic aldehyde stage that was subsequently reacted with hydrazide latex beads to form Schiff base linkages that were reduced with sodium cyanoborohydride. The authors indicated that the oligonucleotide diol was a stable intermediate but that the aldehyde should be prepared immediately before coupling to the latex bead to minimize undesirable reaction of the aldehyde with the oligonucleotide bases.
Another article of special interest as background to the present invention is by Tsarev et al. (Biorg.Khim., 16: 765-79 (1990)) that describes coupling of an aromatic aldehyde to the 5xe2x80x2 phosphate of an oligonucleotide through alkylation. The product was used to probe the RNA polymerase promoter complex.
Typically, glass surfaces are activated by the introduction of amino-, sulfhydryl-, carboxyl- or epoxyl-groups to the glass using the appropriate siloxane reagent. Specifically, immobilization of oligonucleotide arrays on glass supports has been described: by Guo et al., Nuc. Acid Res., 22: 5456-5465 (1994) using 1,4-phenylene diisothiocyanate; by Joos et al., Anal. Biochem., 247: 96-101 (1997) using succinic anhydride and carbodiimide coupling; and by Beatti, et al., Mol. Biotech., 4: 213-225 (1995) using 3-glycidoxypropyltrimethoxysilane.
The rapid specific reaction of cytidine in single stranded DNA with semicarbazide moiety containing reagent, in the presence of bisulfite, has also been described (Hayatsu, Biochem., 15: 2677-2682 (1976)).
Procedures which utilize arrays of immobilized oligonucleotides, such as sequencing by hybridization and array-based analysis of gene expression are known in the art. In these procedures, an ordered array of oligonucleotides of different known sequences is used as a platform for hybridization to one or more test polynucleotides, nucleic acids or nucleic acid populations. Determination of the oligonucleotides which are hybridized and alignment of their known sequences allows reconstruction of the sequence of the test polynucleotide. See, for example, U.S. Pat. Nos. 5,492,806; 5,525,464; 5,556,752; PCT Publications WO 92/10588, WO 96/17957 and the scientific publications by Ramsay, Nat. Biotechnol., 16: 40-4 (1998) and by Lipshutz et al., Nat. Genet., 21: 20-24 (1999)).
Hybridization based DNA screening on peptide nucleic acid (PNA) oligomer arrays has been described (Weiler et al, Nucl. Acids Res., 25: 2792-9 (1997). PNAs and PNA/DNA chimeras are also well described.((Nielsen, Curr Opin Biotechnol. 10: 71-5 (1999); Koch et al., Tetrahedron Let., 36: 6933-6936 (1995)).
However, many of the current immobilization methods suffer from one or more of a number of disadvantages. Some of these are, complex and expensive reaction schemes with low oligonucleotide loading yields, reactive unstable intermediates prone to side reactions and unfavorable hybridization kinetics of the immobilized oligonucleotide. The efficient immobilization of oligonucleotides on glass surface in arrays in a high-through put mode requires a) simple reliable reactions giving reproducible loading for different batches, b) stable reaction intermediates, c) arrays with high loading and fast hybridization rates, d) high temperature stability, e) low cost, f) specific attachment at either the 5xe2x80x2- or 3xe2x80x2-end or at an internal nucleotide and g) low background.
The present invention represents a significant step in the direction of meeting or approaching several of these objectives.
In accordance with the present invention a Schiff base type covalent linkage is formed between a group containing an NH2 moiety and an aromatic aldehyde or ketone to covalently link an oligonucleotide (ODN) to a solid support. The Schiff base type linkage is formed between the solid support and either the 3xe2x80x2, or 5xe2x80x2 end of the ODN, or between the solid support and one or more intermediate nucleotides in the ODN. Alternatively the Schiff base type linkage is located in a combination of these sites. In this regard it should be understood that the Schiff base type covalent linkage may be situated not directly on the solid support or the ODN but on linking groups (linkers) which are themselves covalently attached to the solid support and to the ODN, respectively. Thus, either the solid support or the ODN or both may include a linking group that includes the xe2x80x94NH2 or aromatic aldehyde group which forms the Schiff base type covalent bond to join the ODN to the solid support.
In accordance with one aspect and preferred mode or embodiment of the invention the Schiff base type covalent bond is formed between a semicarbazide group or moiety of the formula Rxe2x80x2xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94NH2, and the aromatic aldehyde moiety of the formula Rxe2x80x3xe2x80x94Qxe2x80x94CHO, preferably a benzaldehyde moiety, where the group Rxe2x80x2 designates either the solid support or the ODN residue including any linker group attached to the solid support or ODN, and where the Rxe2x80x3 designates the other of said solid support or ODN residues including any linker group attached to them. The symbol Q in this formula designates an aromatic ring or a heteroaromatic ring which may have up to three heteroatoms independently selected from N, O and S, and where the aromatic or heteroaromatic ring may itself be substituted with alkyl, alkoxy or halogen groups where the alkyl or alkoxy group preferably has 1 to 6 carbons. The linkage formed between the solid support and the ODN is thus depicted by the formula
Rxe2x80x2xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94Nxe2x95x90CHxe2x80x94Qxe2x80x94Rxe2x80x3
where the symbols have the meaning provided above.
In accordance with still another aspect and preferred mode or embodiment of the invention the semicarbazide moiety is attached to a glass surface, and the benzaldehyde moiety is attached with a linker to the 3xe2x80x2, or to the 5xe2x80x2 end of the ODN, or to one or more nucleotides situated internally in the ODN. The synthetic methodologies to prepare the semicarbazide modified solid support surface and the aromatic aldehyde coupled ODNs comprise still further aspects of the present invention.
Advantages of the solid support ODN conjugates linked together with the above-summarized Schiff base type linkages including an aromatic aldehyde or ketone, and particularly with semicarbazone linkages, include (a) their ability to be formed below pH 7, (b) stability of the Schiff base-with-aromatic-aldehyde bonds and particularly of the semicarbazone-formed-with-an-aromatic-aldehyde bonds, (c) ability to attach a high percentage (typically more than 60%, and preferably about 90%, even more preferably 95% or more) of the ODN to the semicarbazide moiety containing solid support, d) specific attachment at either the 5xe2x80x2- or 3xe2x80x2-end or at an internal nucleotide, and (e) obtaining high coupling densities (preferably of about 104 oligonucleotides/xcexcm2 and most preferably about 105 oligonucleotides/xcexcm2) on unit surface of the solid support. These advantages are to be contrasted with the prior art procedures, see for example [Kremsky et al. (Nuc.Acid Res., 15: 2891-2909 (1987))] where an aliphatic aldehyde attached to the ODN is coupled with a hydrazide-containing solid support to form a hydrazone that is unstable and must be reduced to provide a stable solid support-ODN conjugate.
Another aspect of the present invention is a general method of attaching oligonucleotides to a solid support at a specified end, or other position specified by the placement of the aldehyde or semicarbazide moiety to generate probes for specific polynucleotide sequences. In this case the oligonucleotide is usually attached at the 3xe2x80x2- or 5xe2x80x2-end so that the extent of the oligonucleotide sequence is available for hybridization to a target polynucleotide in, for example, a mixture of polynucleotides of different sequence. The polynucleotides can be labeled with fluorescent, radioactive, chemical or other detectors known in the art. The oligonucleotides may also contain moieties that enhance duplex stabilization, such as minor groove binders, as is described in U.S. Pat. No. 5,801,155, incorporated herein by reference, or modified bases such as pyrazolopyrimidines (as is described in PCT US99/07492 incorporated herein by reference. The stringency and specificity of the hybridization can be adjusted by any of several means known in the art, such as temperature, salt concentration and composition and/or chaotropic agents as is described in the publication by Van Ness et al., Nucleic Acids Res. 19: 5143-51 (1991) incorporated herein by reference, such that only perfectly base-paired duplexes form and the unhybridized polynucleotides removed by washing and the hybridized polynucleotides identified by their attached label(s).
In still another aspect of the present invention, the oligonucleotide attached to a solid support as described above, can be used to hybridize to a polynucleotide, present in a mixture of polynucleotides, under specific conditions, and the unhybridized polynucleotides removed as described above. A second oligonucleotide containing a specific distinguishable label (e.g. radioactive isotope, fluorophore or chemically identifiable compound) can be used to hybridize to a region of the target polynucleotide separate from that hybridized to the first oligonucleotide fixed to the solid support. The second oligonucleotide can contain compounds that enhanced specific duplex formation, such as minor groove binders (U.S. Pat. No. 5,801,155) or modified bases such as pyrazolopyrimidines (PCT Publication US99/07492) and intercalators (as described for example by Gago, Methods, 14: 277-92 (1998) incorporated herein by reference.). After washing to remove unhybridized oligonucleotide(s), the presence of the sequence to which the second oligonucleotide hybridized can be determined by measuring the presence of the label on the second oligonucleotide. Multiple oligonucleotides can be attached to the solid support and multiple oligonucleotides each with a specific label can be used as the second oligonucleotide.
In yet another aspect of this invention, the second oligonucleotide and the first (attached) oligonucleotide can be chosen so that if they hybridize adjacent to one another on the complementary labeled target polynucleotide, they can be ligated to one another, thereby forming a longer oligonucleotide with an inherently higher melting point (Tm). In this case the washing conditions can by adjusted so that no oligonucleotide that is unligated can remain in a duplex with the target polynucleotide. The use of DNA and RNA ligases is well known to those skilled in the art (see for example Lee, Biologicals, 24: 197-199 (1996) incorporated herein by reference).
In a further aspect of this invention, the second labeled oligonucleotide and the first (attached) oligonucleotide can be chosen so that if they hybridize adjacent to one another on the complementary target polynucleotide, they can be ligated to one another, only if they are a perfect match.
In a still further aspect of the invention, oligonucleotides can be constructed with the aldehyde (or semicarbazide) incorporated in one of the described configurations so that the 3xe2x80x2-terminus of the oligonucleotide can be extended by a polymerase. The oligonucleotides so constructed can be used as single primers to generate cDNA or as one member of a primer pair to generate amplicons in the polymerase chain reaction in reactions well described in the literature (see for example Ausubel et al. Edit., in Current Protocols in Molecular Biology, 1:5.5.1-5.5.10 (1990) John Wiley and Sons, New York, incorporated herein by reference). In each of these cases the polynucleotide to be analyzed serves as the template for polynucleotide synthesis primed by aldehyde or semicarbazide containing oligonucleotides which are at least partially complementary to the polynucleotide. The product double-stranded polynucleotide can be attached to a solid support containing the semicarbazide (or aldehyde) moiety. This support containing the double stranded molecules can be used to capture and detect or purify molecules that bind to the sequences present in the attached polynucleotides. Alternatively, the attached double-stranded polynucleotides can be denatured (for example by heating or treating with reagents like NaOH) and the single strand polynucleotides attached via the Shiff""s base covalent linkage remain attached to the solid support and the complementary strand(s) are removed. The single strands remaining on the solid support can now be used as hybridization targets or as targets for other molecules that bind to single stranded polynucleotides.
In a variation of this method, the double stranded polynucleotides can be denatured (i.e. the duplexes converted to single strands) prior to attachment to the solid support via the Schiff""s base formation.
In yet another aspect of this invention, an oligonucleotide can be constructed so that attachment to the solid support via the Schiff""s base is achieved so that the oligonucleotide is free to hybridize to a target polynucleotide so that it can be digested by an enzyme that acts only on double strand polynucleotides. The attached oligonucleotide is constructed to contain a fluorophore and a quencher that blocks the fluorescence of the fluorophore, positioned in such a way that when the oligonucleotide forms a duplex with its complement in a mixture of polynucleotides, it can be cleaved by the enzyme to separate the quencher and the fluororphore, generating a fluorescent signal at the position on the solid support where the oligonucleotide was attached. Although many different double strand specific enzymes would be useful in this method, a specific case would be the use of DNA polymerases in the polymerase chain reaction. In this case the oligonucleotide attached to the solid support contains a quencher at its 5xe2x80x2-end and a fluorophore coupled elsewhere, usually at the 3xe2x80x2-end. A second oligonucleotide complementary to the target polynucleotide in a region serves as a primer 5xe2x80x2 from the attached oligonucleotide (containing the quencher and fluorophore). As the polymerase extends the primer, it digests the attached oligonucleotide from the 5xe2x80x2-end releasing the base(s) to which the quencher is attached and the polynucleotide-attached oligonucleotide duplex dissociates, leaving the portion of the attached oligonucleotide containing the fluorophore attached to the solid support. Subjecting the resulting oligonucleotide to the appropriate light generates a fluorescent signal at the position of the attachment of the oligonucleotide. FIG. 6 shows where in addition to the fluorophore and quencher, a minor groove binder is also incorporated into the oligonucleotide conjugate. Reagents and methods for carrying out polymerase chain reactions on solid supports are well known to those skilled in the art, see for example Cheng et al., Nucl. Acids Res. 24: 380-385 (1996).
Another aspect of the present invention is a general method for the isolation of single stranded DNA in a process where an aldehyde-labeled primer is used and an amplicon is immobilized on a semicarbazide containing solid support. Denaturation of the amplicon and separation yield single stranded DNA in solution and on the solid support, which could be used individually for many applications known in the art. This is an improvement and further development of the procedure described by Mitchell et al., Anal. Biochem., 178: 239-42 (1989), where single-stranded DNA is xe2x80x9caffinity generatedxe2x80x9d following a polymerase chain reaction using a biotinylated primer, followed by streptavidin-solid support separation.
In accordance with yet another aspect of the present invention, non-specific adsorption of the negatively charged nucleic acids to the semicarbazide or other amine-modified glass surface can be largely eliminated by converting the unreacted NH2 groups (preferably semicarbazide xe2x80x94Rxe2x80x2xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94NH2 groups) into a moiety containing an anion. This is accomplished by reacting the ODN attached to the solid support with a reagent that introduces an anionic group, for example by reacting the solid support with 4-formyl-1,3-benzenedisulfonic acid. In addition, unreacted silanol functions on the solid support, preferably glass surface are end-capped with a hydrophobic siloxane to increase stability of the immobilized oligonucleotides.
Although this is not usually necessary, the semicarbazone linkages formed with the aromatic aldehyde moiety and linking the oligonucleotide with the solid support can be reduced to provide still stable solid-support-ODN conjugates.
In accordance with a still further aspect of the present invention an ODN containing cytidine is immobilized on a solid support containing semicarbazide groups by bisulfite catalyzed covalent attachment through the cytidine nucleotides of the ODN.
It should be understood that generally speaking for the purpose of this invention an oligonucleotide comprises a plurality of nucleotide units, a 3xe2x80x2 end and a 5xe2x80x2 end. The nucleotide may contain one or modified bases other than the normal purine and pyrimidine bases. In addition an oligonucleotide may include peptide oligonucleotides (PNAs) or PNA/DNA chimeras. The oligonucleotide may also contain groups that can influence its binding to a complementary strand, e.g. minor group binders or intercalators, or groups necessary for its detection e.g. fluorophores and quenchers
The present invention is primarily used at present for the capture and detection of nucleic acids using oligonucleotides attached to glass surfaces with the Schiff base type, (preferably semicarbazone) bonds, and more particularly for the capture and detection of PCR generated nucleic acid sequence in array format, although the use of the invention is not limited in this manner. Generally speaking the oligonucleotides immobilized on solid support in accordance with the present invention exhibit superior direct capture ability for complementary oligonucleotide, DNA and RNA sequences.